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. 2023 Apr 11;13:5891. doi: 10.1038/s41598-023-32720-3

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Immunostaining for pro-NPFF in a section obtained from a NPFF^Cre;GRPR^Flp mouse that had been injected with AAVs coding for Cre-dependent GFP and Flp-dependent mCherry. (a–c) show staining for GFP (green), mCherry (red) and pro-NPFF (blue) in a single confocal optical section, while (d) shows a merged image. The arrow and asterisk mark cells that are positive for GFP and negative for mCherry. The cell indicated with the asterisk contains pro-NPFF, and this is seen more clearly in a different optical section (inset). The cell marked with the arrow lacks pro-NPFF. The cells indicated with single and double arrowheads are both positive for mCherry. One of them (double arrowheads) is also labelled with GFP, while the other (single arrowhead) is not. Both of these cells lack pro-NPFF-immunoreactivity. (e) Venn diagram showing the relationship between cells labelled with each fluorescent protein and pro-NPFF immunoreactivity. pro-NPFF was present in 0.4% of GRPR cells (labelled with mCherry), 25% of NPFF cells (labelled with GFP) and 42% of the cells that were GFP-positive/mCherry-negative. Scale bar = 20 μm.